[unreadable] The objective of this project is to determine if the enzyme cyanuric acid amidohydroase (encoded by genes termed atzD or trzD) can serve as a selectable biomarker in pathogenic Burkholderia. The Specific Aims are: 1.) To determine if expression of atzD/trzD in Burkholderia mallei confers growth on CA as a sole N source, 2.) To develop a broad host range vector that utilizes atzD/trzD as a selectable marker in Burkholderia mallei, 3.) To develop a suicide vector system that utilizes atzD/trzD as a marker for mutating targeted genes in B. mallei, 4.) To utilize the atzD/trzD-suicide vector to construct isogenic mutations in a B. mallei ATCC 23344 gene encoding a putative adherence factor, and 5.) To test the utility of atzD/trzD as a biomarker in B. pseudomallei. Burkholderia mallei and Burkholderia pseudomallei are human pathogens and the causative agents of glanders disease and melioidosis, respectively. Both organisms are agents of biodefense concern, and consequently there is great interest in understanding the mechanisms underlying their pathogenicity. Molecular genetics approaches are key to such studies, but are hampered by the limited availability of suitable selectable markers. Preliminary tests of non-pathogenic soil isolates of Burkholderia sp. and B. mallei ATCC 23344 (the model organism used in studies on B. mallei biology) have shown these organisms possess a physiological background that would allow the introduction and expression of a trzD/atzD homolog as a selectable biomarker. The proposed studies will focus on development of atzD/trzD as a biomarker B. mallei ATCC 2344. A secondary goal will be to examine the utility of trzD/atzD as a biomarker in B. pseudomallei. Initial testing of the testing of the trzD/atzD biomarker will be done in non-pathogenic soil isolates of Burkholderia sp. Final testing will be done in B. mallei ATCC 2344 and B. pseudomallei strain DD503. Use of CA as a biomarker would have several advantages. First, unlike almost all traditional biomarkers, it utilizes a nontoxic compound that poses no environmental or personal safety hazards. Second, selection would be based on use of CA to support growth rather than resistance to an antimicrobial agent; thus there are no concerns about the selection leading to dissemination or resistance determinants. Third, the marker could be used alone, or in combination with other markers, which could provide greater flexibility in molecular genetics studies. [unreadable] [unreadable] [unreadable]